Anti rabbit igg fab2 alexa fluor r 488

The non-polarized and polarized hEHs were fixed using 4% paraformaldehyde fix solution. After permeabilizing by 0.5% Triton and blocking by goat serum, the cells were incubated with primary antibody at 4 °C overnight. Then, the cells were co-incubated with anti-rabbit IgG Fab2 Alexa Fluor (R) 488 (Cell Signaling Technology, Boston, MA, USA) and Phalloidin-iFluor 594 (Abcam, Cambridge, UK). After washing, the cells were incubated with DAPI staining solution (Beyotime). After being thoroughly washed, the samples were removed from the hanging insert and placed on microscope slides. The fluorescence signal was imaged on the single photon confocal microscopy (Ti-E A1, Nikon, Tokyo Met. Japan). Antibodies used were listed in Table S1.

In total, 1 × 10⁵ HER2-overexpressing breast cancer cells (SK-BR3, BT474, MDA-MB361) and HER2 low-expressing breast cancer cells (MDA-MB231, MDA-MB468) were seeded on coverslips at the bottoms of wells in a 24 well-plate. Cells were fixed in 4% paraformaldehyde for 15 min, washed with phosphate-buffered saline (PBS) twice, and non-specific sites were blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Cells were incubated with anti-HER2 antibody (HER2/ErbB2 (D8F12) XP^TM Rabbit mAb, Cell Signaling technology, Danvers, MA, USA) overnight at 4 °C, followed by incubation with the appropriate Alexa Fluor 488 secondary antibody (1:1000, Anti-Rabbit IgG Fab2, Alexa Fluor (R) 488, Cell Signaling technology) for 1 h at room temperature. The cover slips were added the mounting medium with DAPI for 10 min prior to imaging by fluorescence microscope (LSM 700 confocal, ZEISS, Oberkochen, Germany).